A. Field of the Invention
This invention relates to the treatment of harmful inflammation in body tissue by exposing the inflammatory cells to heat. In one method, heat is applied to an inflamed atherosclerotic plaque by means of a catheter that is equipped with a heating source. Such heating can be very mild (38° C.–40° C.) so as to quiesce macrophages and other inflammatory cells, or moderate (41°–44° C.) to induce programmed cell death, apoptosis, but not hot enough to cause necrosis and subsequent inflammation.
B. Description of Related Art
Coronary artery disease is a leading cause of death in industrialized countries. It is manifested by athersclerotic plaques, which are thickened areas in vessel walls. A plaque is an accumulation of cholesterol, proliferating smooth muscle cells and inflammatory cells covered by cellular secretions of collagen that form a cap over the plaque in the vessel wall. Macrophages migrate into and accumulate in a plaque causing inflammation. Inflamed plaques are most susceptible to ruptures and the formation of blood clots. Falk, E. (1995).
Atherosclerotic plaques are thought to develop in response to irritation or biochemical damage of the endothelial cells that line blood vessel walls. Agents that are known to damage these cells include cigarette smoke, high serum cholesterol (especially in the form of oxidized low density lipoprotein), hemodynamic alterations (such as those found at vessel branch points), some viruses (herpes simplex, cytomegalovirus) or bacteria (e.g., Chlamydia), hypertension, and some plasma hormones (including angiotensisn II, norepinephrine) and homocysteine. Atherosclerotic plaques grow slowly over many years in response to the cumulative injury of endothelial cells. Ross (1993), Berliner (1995).
Typically, several dozen plaques are found in arteries afflicted with this disease. It is the rupture of these plaques that brings about the terminal stage of the disease. The rupture causes a large thrombus (blood clot) to form on the inside of the artery, which may completely occlude the blood flow through the artery, thereby injuring the heart or brain. Falk, E. (1995).
In most cases of terminal coronary artery disease, only one of several plaques ruptures. Rupture typically is caused by inflammatory cells, primarily macrophages, that lay beneath the surface collagen layer of the plaques. These cells release enzymes that tend to degrade the cap. Once a plaque ruptures, blood clots are formed and it is these clots that are believed to be responsible for over one half of all heart attacks and stokes. Falk, E. (1995); Buja (1994).
Techniques have been developed to identify those plaques that are most likely to rupture because of inflammation. See U.S. patent application Ser. No. 08/717,449, which is specifically incorporated by reference herein. The most common treatment for these plaques is balloon angioplasty. However, this method is known to result in about a 5% incidence of complete thrombotic occlusion and a 30–40% incidence of vessel reclosure due to restenosis. It is clear that in many cases balloon angioplasty causes cellular injury and only temporarily eliminates the danger from an inflamed plaque until the advent of a secondary inflammatory response. Casscells (1994).
It has been shown that macrophages have a life span of only about a week or two in the vessel wall. Katsuda (1993). Typically, monocytes enter the atherosclerotic plaque, divide once, and contribute to plaque development by their ability to oxidize low density lipoprotein cholesterol and to release factors which cause smooth muscle proliferation and angiogenesis. The cells then undergo apoptosis, which is an active process of programmed cell death. This process differs from necrosis in that apoptosis requires the expenditure of energy, and the synthesis of new RNA and proteins in all but the inflammatory cells, the active cleavage of DNA and the shrinkage and involution of the cell with very little inflammation. Steller (1995); Nagata (1995); Thompson (1995); Vaux (1996).
Apoptosis is a form of programmed cell death in which the dying cells retain membrane integrity and are rapidly phagocytosed and digested by macrophages or by neighboring cells. It occurs by means of an intrinsic cellular suicide program that results in DNA fragmentation and nuclear and cytoplasmic condensation. The dead cells are rapidly cleared without leaking their contents and therefore little inflammatory reaction follows. It can be induced by the withdrawal of growth factors and to some extent by factors which can also cause necrosis such as extreme lack of oxygen or glucose, heat, oxidation and other physical factors.
Previously no method was known for selectively inducing apoptosis in macrophages or other inflammatory cells in a blood vessel without also inducing apoptosis in beneficial endothelial cells. Known methods for inducing apoptosis were systemic, including treatments with chemicals and elevated temperatures. Such methods are not useful as therapeutic methods because of the risk that apoptosis will develop in healthy tissue.
A number of studies have shown that heat can induce programmed cell death. Kunkel (1986) have found that indomethacin inhibits macrophage synthesis of prostaglandin but enhances macrophage production of TNF-α, which suggests that heating may have advantages over indomethacin as an anti-inflammatory treatment. Preventing the synthesis of prostaglandin, which serve as feedback inhibitors of macrophage function, limits the anti-inflammatory utility of indomethacin and presumably other inhibitors of cyclo-oxygenase. Field and Morris (1983) surveyed many cell types and found that the time needed to kill cells at 43° C. varied from four minutes in mouse testis, to 32 minutes in rat tumor 96 in vivo, to 37 minutes for mouse jejunum, to 75 minutes for rat skin, 210 minutes for mouse skin and 850 minutes for pig skin. Numerous other cell types were also studied. They observed that, above 42.5° C., an increase of 1° C. produces a similar effect as doubling the duration of heat exposure. Wike-Hooly (1984) found that a low pH enhanced hyperthermic cell killing, as did a low glucose or insulin exposure and that nitroprusside also increased the cell mortality caused by hyperthermia. Raaphorst (1985) and Belli (1988) studied Chinese hamster lung fibroblasts and found that 45° C. heat and radiation were synergistic in cell killing. Raaphorst (1985) also found S-phase to be heat-sensitive and least radiosensitive, while in G1 and G2 the opposite was true. Klostergard (1989) found that cytotoxicity of macrophages was decreased by heating to 40.5° C. for 60 minutes. Westra and Dewey (1971) found that in CHO cells S phase was more sensitive to heating to 45.5° C. than was G1 phase. M phase was intermediate. In contrast, radiation killed cells preferentially in phases G1 and M1. Fifty percent of asynchronous (cycling) CHO cells were killed by a 20 minute heat treatment at 43.5° C. Freeman (1980) found that the sensitivity of CHO cells to 41° C. to 45.5° C. was increased with acidosis and that thermotolerance was induced by exposure to 42° C. for 250 minutes. Haverman and Hahn (1982) used an inhibitor of oxidative phosphorylation and found that CHO cells were thereby more prone to heat-induced death using 43° C. for one hour. Preheating, however, led to tolerance. These experiments could not determine whether hyperthermia increased ATP utilization or inhibited its synthesis. Gerweck (1984) found that CHO cells were more easily killed by 44° C. (20% died after a 15-minute exposure) when ATP was depleted by hypoxia and hypoglycemia, but neither condition alone had an effect. Lavie (1992) found that peritoneal macrophages from older mice tend to die at 42.5° C. for 20 minutes but not macrophages from younger mice. Papdimitriou (1993) found that most peritoneal murine macrophages undergo apoptosis with a five-hour exposure to 41° C., but few entered apoptosis at 30° C. Most circulating monocytes did not undergo apoptosis at 41° C., with a five-hour exposure. Mangan (1991) reported that TNF alpha and interleukin-1 beta prevent macrophage apoptosis. Chen (1987) reported that heat in the range of 41° C. to 43° C. stimulated macrophage production of prostaglandins. Prostaglandins serve to suppress macrophage production and phagocytosis. Heat did not decrease prostaglandin release from tumor cell line or from fibroblasts. They found that macrophage death began at 41° C. with a four-hour exposure. A six-hour exposure to 43° C. killed half the macrophages. Ensor (1995) found no macrophage cell death after six hours at 40° C., (vs. 37° C.) but at 43° C. only 4% of cells were viable at six hours. O'Hara (1992) found that bone marrow macrophages survive 15 minutes at 45° C. if they have been preheated for 110 minutes to 42.5° C. Fouqueray (1992) found that exposing rat peritoneal macrophages to 39° C. to 41° C. for 20 minutes decreased synthesis of IL-1 and TNF-α. Circulating monocytes were less sensitive to heat than glomerular or peritoneal macrophages. This degree of heating did not kill the macrophages. Hamilton (1995) found that the cancer drug bleomycin blocked expression of HSP-72 in human alveolar macrophages in response to exposure to 39.8° C. This was a relatively specific effect since there was no change in overall protein synthesis and, moreover, the effect appeared to be post-transcriptional, since there was no change in mRNA levels for HSP-72. The bleomycin exposure did not cause much necrosis, but it caused marked DNA fragmentation characteristic of apoptosis. Wang (1995) found that induction of HSP-72 prevented necrosis in human endothelial cells exposed to activated neutrophils. The activated neutrophils caused necrosis of endothelial cells that had been exposed to 30 to 60 minutes of heat shock at 42° C., an exposure which by itself did not induce necrosis or apoptosis. Wang (1997) found that endothelial cells did not go to apoptosis with a 45-minute exposure to 42° C. or with exposure to TNF-α, but exposure to both did trigger apoptosis. TNF-α resulted in generation of reactive oxygen species, which the authors believe may be required, together with heat shock, to induce apoptosis in endothelial cells. Kim (1997) found that nitric oxide protected cultured rat hepatocytes from TNF-α induced apoptosis by means of inducing HSP-70. Belli (1963) observed that heating enhances cell susceptibility to radiation killing.
Cytokines are also known to influence apoptosis in macrophages and other leukocytes. William (1996) found that apoptosis in neutrophils was promoted by heat, TNF-α, or endotoxin but inhibited by LPS, GMCSF and IL-2. Biffl (1996) found that IL-6 also delayed neutrophil apoptosis. Prins (1994) found in human fat tissue explants adipocytes underwent apoptosis within 24 hours of a 60-minute exposure to 43° C. and then underwent phagocytosis, suggesting that at least some macrophages survived longer than some adipocytes. O'Hara (1992) showed that granulocyte-macrophage precursors take longer (T1/2=36 min.) to become heat-tolerant than do stem cells or erythrocyte precursors from bone marrow. Verhelj (1996) found that 50 percent of confluent, nondividing, bovine aortic endothelial cells underwent apoptosis by 12 hours at 43° C., versus 41° C. for dividing human monoblastic leukemia of the U937 line, but this difference could well be attributable to the difference in age of the cells, cycling rate or species. D. Elkon and H. E. McGrath (1981) presented some evidence that granulocyte monocyte stem cells do not take as long as other cells to be killed at a temperature of 42.5° C. Blackburn (1984) reported that circulating monocyte precursors are more sensitive to heat than are those from bone marrow. Kobayashi (1985) reported that granulocyte-macrophage progenitor cells were more sensitive to 60 minutes at 42° C. when the marrow was regenerating (during cell division) than when it was stationary, but this is a finding in all cell types. Cohen (1991) found no difference in heat tolerance of epithelial cells and airway macrophages, as measured by immediate release of LDH and chromium-51.
A number of studies have studied the relationship between heat shock and cell killing. Nishina (1997) found that the stress-activated protein kinases (also known as the Jun N-terminal kinases) are activated in response to heat shock and other cell stresses. A knockout of one of these genes (SEK-1) resulted in fewer CD4+, CD8+ thymocytes. Pizurki and Polla (1994) found that cAMP increased synthesis of heat-shock proteins by heated macrophages. Reddy (1982) found that heat shock of murine macrophages increased their production of superoxide but did not change their production of hydrogen peroxide or their microbicidal activity. Sivo (1996) found that heat shock acted in a fashion similar to glucocorticoids in inhibiting mouse peritoneal macrophages and increased the transfer of glucocorticoid receptors to the nucleus. Snyder et. al (1992) found that mouse peritoneal macrophages synthesized heat-shock proteins (HSPs) maximally after a 12-minute exposure to 45° C.; HSPs were only found two to six hours after heat treatment. They found no HSP-70 at 42° C. or 43° C. At 2 and 24 hours after heating, phagocytosis was normal. They did not mention whether macrophages entered apoptosis with this treatment and that the same treatment (12 minutes at 45° C.) decreased TNF alpha and IL-1 RNA synthesis in mouse peritoneal macrophages. Pizurki et. al (1994) reported that circulating human monocytes express HSPs two hours after 20 minutes of exposure of 45° C. and that expression was enhanced in cAMP and unaffected by indomethacin.
A number of studies have shown that heating and chemical treatments change the activity of immune cells. Chen (1987) found that heating murine macrophages to 41° C. to 43° C. for one hour caused them to synthesize and release prostaglandins of the E type. Fouqueray (1992) found that a 20-minute exposure of rat peritoneal macrophages to 39° C. to 41° C. decreased synthesis of tumor necrosis factor alpha and interleukin-1 within two hours, but monocytes circulating in the blood were less sensitive to heating than were the tissue macrophages. Ribeiro (1996) confirmed that heat exposure decreases macrophage release of TNF alpha both in vitro and in vivo. Kunkel (1986) showed that indomethacin inhibited lipopolysaccharide (LPS)-induced synthesis of prostaglandins by macrophages (and inhibited heat-induced PGEs Chen, 1987) but enhanced macrophage production of TNF-α in response to LPS. Morange M. (1986) found that HSPs were induced at lower temperatures when cells were exposed to interferon-alpha and interferon-beta. Ensor (1995) reported that exposing a macrophage cell line to 40° C. for 30 minutes prevented (within six hours) synthesis and release of TNF-α in response exposure to LPS. The half-life of TNF-α mRNA was shortened. There was no change in the levels of mRNA for GAPDH, β-actin or IL-6. HSP-72 was increased at 43° C. The same authors previously showed that in human macrophages TNF expression was suppressed at 38.5° C., but HSP-72 was increased only above 40° C. Papadimitriou showed that macrophage apoptosis was minimal at 39° C. but substantial at 41° C.
Although the cellular phenomenon of apoptosis has been studied in some detail in tissue culture, no studies have been directed toward developing that technique for the treatment of inflammation. New methods are needed for treating inflamed body tissues and in particular to the treatment of atherosclerotic plaques to prevent rupture. Such methods should not induce an inflammatory response and should be capable of eliminating or neutralizing macrophages or other inflammatory cells without damaging blood vessel walls. Novel methods for selectively inducing apoptosis are also needed. Such methods will be useful in preventing the rupture of atherosclerotic plaques and therefore reduce the risk of death from myocardial infarction or stroke.